工程化组织的构建.pptx

1、四川大学华西临床医学院 干细胞与组织工程研究室 秦廷武,Dec.22,2006,工程化组织的构建(Construct of engineered-tissues),The concept of tissue engineering,The term tissue engineering was officially coined at a National Science Foundation workshop in 1988 to mean the application of principles and methods of engineering and life sciences t

2、oward fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore,maintain or improve tissue function.,Purposes of tissue engineering,To develop biological substitutes forimplantation into the bod

3、y;To foster tissue regeneration and remodeling;To replace,repair,maintain,orenhance function of tissues.,Medical application purposeResearch purpose,Testing new drugs;Simulating diseases to develop better treatments;Reducing the use of animal tissues and organs in biological research.,组织工程研究内容,组织工程研

4、究过程,工程化 组织,构建模式 体内 体外,细胞 改造,材料设计 加工检测,生物学及 力学检测,植入技术修复技术,细 胞 接 种 应力与生长,生物反应器、力学 诱导与信号的转导,种子细胞,干细胞 工程,支架材料,工程化组织的保存,植入技术 修复技术,Simple culture techniques,If specific signals are not properly received or processed,then the cells dedifferentiate(i.e.,become nonspecific cell types),become disorganized,and

5、 eventually die.,Dynamic microenvironment,Cell colonies need external cues or signals to grow into functional 3D tissues.Cells are constantly bombarded with mechanical,electrical,structural,and chemical cues that signal the cells about what they should be doing.,工程化组织的理解,Special culture,Engineered t

6、issue,The engineered tissue，or construct，usually consists of three-dimensional scaffolds and living cells with the desired structure and functionality.,细胞材料有机结合体,细胞,材料,？,Bioreactors for Tissue Culture,Bioreactors are purpose-designed tissue culture system；Providing the desired biochemical and mechan

7、ical environment for cell adhesion,proliferation,differentiation and function；Devising and developing new types of bioreactors,particularly addressing mass transfer and mechanical signaling.,简单,复杂,低维,多维,On-line monitoring in engineered tissue,Monitoring of the cell viability,functionality and tissue

8、 formation；Developing continuous sampling and analytical technologies using micro-or nano-probes；Enabling efficient control of the tissue culture bioreactor and optimization of the construct design and bio-processing operation.,Cryopreservation of engineered tissues,Owing to the long production cycl

9、e,for example three weeks for engineered skin grafts to eight weeks for engineeredcartilage,preservation of engineered tissues is critical to ensure the off-the-shelf availability to clinicians.Preservation and storage of live engineered tissues,or bio-constructs,would be one of the major obstacles

10、for the commercialization.,Mathematical modeling in tissue engineering,Developing mathematical models for cell growth and tissue formation；Devising experimental techniques to determine model parameters such as oxygen consumption rate and effective diffusivity in the tissue；The models can be applied

11、both in vivo and in vitro.,工程化组织的构建方法,体外构建（工程化椎间盘）,体内构建（工程化耳软骨）,体外构建,静态:细胞或支架相对静止；动态:细胞或支架相对运动；存在的问题：环境条件、营养条件(生物反应器)。,在体外模拟体内细胞的动态微环境，包括 生物化学、生物物理环境。提供组织特有的生 物的、化学的和物理的刺激，产生相应的传导 信号。并提供细胞营养及代谢通路。,生物反应器系统,静态:常规二氧化碳培养箱；动态:旋转培养系统、拉伸/压 缩/扭转培养系统、电磁培养 系统、声学/光学培养系统。,Perfusion of Culture System,Improve mas

12、s transport throughout the scaffold by perfusing media through cell seeded scaffolds and stimulate those cells via flow-induced shear stress.,Constructs are perfused using a peristaltic pump at varying flow rates to enhance matrix mineralization.,体外构建人工皮,Engineered skin constructs in vitro keratinoc

13、ytes growing on a plasma polymer surface,SEM,体外构建的耳软骨,Newly formed in vitro auricular cartilage construct,Ear cartilage construct with the chondrocytes,动态构建:有软骨陷窝;有软骨基质分泌.静态构建:无软骨陷窝;无软骨基质分泌.,静态、动态构建的差异（软骨）,本位：功能位置；皮下：浅表位置；肌内：深度位置；骨内：骨膜内。,体内构建,体内构建的皮肤,Reconstructed human skin on the dorsal of the nud

14、e mice at 4 weeks after transplantation,体内构建气管软骨,皮下：浅表位置,皮下构建气管软骨,体内、外构建的差异,In vitro constructs formed and maintained at 8 weeks:Yellowish glistening surface；Resembling native cartilage；Stable but soft in consistency.,In vivo constructs after maturation in mice：Whitish with glistening；Firm in consis

15、tency.,体内、外构建的差异（关节软骨）,Articular cartilage constructs in vitro,Articular cartilage constructs in vivo,体内、外构建的差异（皮肤）,Apligraf consists of living cells and structural proteins:Lower dermal layer combines type 1collagen and fibroblasts(dermal cells),which produce additional matrix proteins;Upper epider

16、mal layer is formed bypromoting human keratinocytes(epidermal cells)to multiply and to differentiate to replicate the architecture of the epidermis.,Apligraf without:Melanocytes;Macrophages;Lymphocytes;Blood vessels;Hair follicles;Sweat glands.,工程化组织的检测,检测对象：细胞、支架、工程化组织及植 入后新生组织;检测指标：形态学、组织学、分子生物学 及

17、生物力学。,形态学检测,光学显微镜；电子显微镜；荧光显微镜；激光共聚焦显微镜；原子力显微镜。,组织学检测,固定、包埋；切片（软、硬、冰冻）；染色（免疫组织化学：因不同组织而定）；荧光或同位素标记；测量（半定量、定量）。,生物力学检测,样品制作、保存；实验设计：选择指标、样本量、数据处 理及统计方法；静态实验（拉伸、压缩、剪切、弯曲、扭转）；动态实验（疲劳、磨损、寿命）。,生物力学检测指标,强度指标：拉伸强度、压缩强度、弯曲强度、扭转强度、剪 切强度和疲劳强度；刚度指标：扬氏模量、剪切模量、弯曲模量、扭转模量等；塑性指标：延伸率、最大应变、断面收缩率、粘性系数等。,工程化肌腱的静态构建,体外静态

18、构建举例,种子细胞与支架材料,种子细胞：第2-4代鸡腱细胞,支架材料：用缝线复合的PGA网,细胞接种及培养,细胞接种:接种密度1106/支架),细胞-支架复合物;静态培养时间:6天;培养条件：37，5%CO2，饱和湿度。,37，5%CO2,细胞-支架复合物,细胞接种工程化肌腱,静态构建的工程化肌腱,腱细胞长入支架中,沿纤维方向平行排列,并分泌胶原基质,组织学检测(HE staining),2W4W,6W,8W,组织学检测（Mallory staining）,2W4W,6W,8W,支架材料的降解,2 week,4 week,6 week,8 week,454035302520151050,PGA

19、支架TET2周4周6周8周,材料重量(mg),支架材料降解速率,称重法,植入前后抗拉强度,120100806040200,支架,TET,2周,4周,6周,8周正常肌腱,抗拉强度(MPa),单轴拉伸实验：每个时间点5个样本;加载速度:20mm/min;应力下降80%停止加载。,植入后胶原生成量,1000,300200,400,2周,4周,6周,8周,正常肌腱,羟脯氨酸(u g/g D ry W t.),*,*,碱水解法测羟脯氨酸,静态构建的结论,静态培养,无体内肌腱细胞特有的力学刺激;支架降解过快;植入后力学刺激小,粘连形成多;胶原合成少,且排列方向无明显取向性;新生腱生物力学强度差.?!?,工

20、程化肌腱的体外动态构建,3-DCultureofTendonCellsunder Mechanical Strain,细胞特性,生物学特性：形态学：正常贴壁依赖性成纤维样肌腱细胞；致瘤性：不致瘤；遗传性状：二倍体细胞；结构：正常细胞结构；功能：能分泌型胶原。力学特性：变形性；黏附性；,3-D Scaffold,3-D scaffold,2g/ml CNI,5g/ml FN,PLGA 85/15,PVAfoam,支架材料形态,大体,表面微观（SEM）,支架材料的力学特性,0,2,4681012Strain(100%),S tre ss(M P a),PVA+PLGA21.510.50,Cell

21、seeding In 3-D scaffold,3-D Scaffold,Cell-Scaffold Composite,Cell Suspension(2105/ml),Seeding twice!,Strain culture device,中国发明专利号:ZL98121829.6,Mechanical strain pattern,10%elongation;12 cycles/min;15 min/h48h;48 hrs.,应变场生物反应器,Scanning electron photomicrographs,静态组,动态组,组织学检测,H.E.染色,静态组（48h,100）,动态组（

22、48h,100）,Proliferation response,210,3,4,HETCs,THETCs,Tendon cellProliferation is increased under mechanical strain,Cell number（106/sponge）,ControlStrain,*,DNA synthesis,When measuring continuous 3H thymidine incorporation into DNA it took 48h to demonstrate stretch-mediated enhancement of DNA synthe

23、sis,*,*,32.521.510.50,HETCsTHETCsTendon cell,3H-TdR incorporation(cpm103/sponge),Control,Strain,Collagen synthesis,0.40,0.8,1.61.2,HETCs,THETCs,肌腱细胞Collagen synthesis is significantly different in stretched culture,compared with nonstretched control culture,3H-Proline掺入量(cpm104/支架),Control Strain,*,

24、*,Cells became more active,and spread to the direction of the strain.After a 48-hour exposure to the strain,cell number DNA synthesis were increased compared with controls.The cyclic strain also caused an increase in collagen matrix synthesis by tendon cells.Cyclic mechanical strains acted directly

25、to stimulate the growth of tendon cells.These results are compatible with a significant role for strain in the construct of engineered tendons in vitro.,动态构建的结论,Many challenges,Missing information on how to develop universal donor cells that could be given to any recipient；How to stimulate regenerat

26、ion of complex multicellular structures in vivo；How to direct the function of its cells；How to vascularize the engineered tissues;Success in tissue engineering is going to require interdisciplinary participation and determination.,Multi-disciplinary cooperation,Biologists source and grow the cells t

27、hat will be used to construct the engineered tissue/organ;Chemists develop a suitable scaffold on which the cells can be grown;Engineers fabricate the scaffold and ensure it has the appropriate physical characteristics;Biochemists and physiologists test cells grown on the scaffold to ensure that they function correctly;Clinicians implant and test the organ/tissue in patients.,